Project Summary Telomerase is a specialized reverse transcriptase (RT) that synthesizes telomere DNA repeats at chromosome ends, using only a very short region of its intrinsic telomerase RNA (TR) subunit as template. This highly specialized function of telomerase relies on a special mechanism whereby the template RNA and the telomeric DNA dissociate and realign during the processive synthesis of repeats. However, the detailed mechanism of telomerase template translocation remains to be determined. This research program aims to articulate the unique mechanism of telomerase action and identify elements that regulate specific steps of template translocation. Although telomerase uses the single-stranded telomeric DNA as its native substrate, we have recently discovered that telomerase can act as a conventional RT utilizing RNA/DNA duplex as substrate. More surprisingly, telomerase recognizes the duplex substrate with a sequence-specificity. These crucial findings have provided great insights into the molecular mechanism of telomerase action. We hypothesize that duplex- binding and duplex-dissociation are important steps of the telomere-repeat synthesis cycle, and regulate telomere-repeat addition rate and processivity. Specific Aims of the research program include (1) Determining the role duplex-binding affinity in template translocation efficiency, (2) Determining the rate-limiting step of template translocation, and (3) characterize the sequence- dependent termination of nucleotide addition by telomerase. We expect the outcomes of these experiments will greatly add to our understanding of telomerase mechanism.